In vivo, net matrix degradation is a function of the balanced activity of proteolytic enzymes and their endogenous inhibitors, in particular the matrix metalloproteinases (MMPs) and their inhibitors, the TIMPs (Tissue Inhibitors of MetalloProteinases). Matrix degradation is an impotant prerequisite of angiogenesis, tunorigenesis and metastasis. This proposal is based on th hypothesis that shifts in the proteolytic balance favoring either MMPs or TIMPs, may have important and clinically relevant effects on the regulation of angiogenesis. Very recently, MMPs have been implicated in the processing of precursor/parent proteins into their bioactive components. Within the context of Specific Aim 1, we will study the role that shifts in the proteolytic balance between MMPs and TIMPs may have in the processing of angiogenesis inhibitors which are fragments of larger, inactive proteins, in particular angiostatin and endostatin, two recently discovered inhibitors. This will be accomplished through the use of substrate fel electrophoresis, radiometric MMP assays, and Western and Northern blot analyses complemented by a series of sense and antisense transfection studies of the relevant MMPs and TIMPs. There studies will be followed by subsequent in vivo bioassays to assess the phenotypic consequences of these genetic modifications. New and interesting functions are being attributed to the TIMPs as well. In addition to their MMP inhibitory activity, some of these inhibitors also possess angiogenesis-doculating activities. Although much is known with respect to the identification of the TIMP domains that are important for MMP inhibition, relatively nothing is known about the TIMP domain(s) responsible for their effects on angiogenesis. Within the context of Specific Aim 2, we will use a yeast expression system to express different structural domains of TIMPs-1 and -2. These domains will then be tested in a series of angiogenesis assays, both in vitro and in vivo, in order to identify the structural determinants of the angiogenesis-modulating activities of the TIMPs.